Pramanik S K, Bag J
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Eur J Biochem. 1989 Jul 1;182(3):687-98. doi: 10.1111/j.1432-1033.1989.tb14880.x.
Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non-proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle-specific genes in mitogen-poor differentiation medium. In the absence of any detectable accumulation of muscle-specific alpha-actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle-specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA-treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non-translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle-specific mRNAs. The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA-dependent micrococcal-nuclease-treated homologous cell-free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing these ribosomes with a high-ionic-strength buffer. In order to elucidate the role of interaction between mRNA and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA-protein (mRNP) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of the RNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein than polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNP complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP.
研究发现，大鼠L6细胞中一种40kDa管家多肽（P40）的mRNA翻译在多种条件下受到调控。这种mRNA在增殖的成肌细胞中可被翻译，但在非增殖的肌管中则不能。使用了多种化学物质，如二甲基亚砜、丁酸钠和阿非迪霉素，来阻止在有丝分裂原含量低的分化培养基中肌肉特异性基因的表达。在没有检测到肌肉特异性α-肌动蛋白mRNA积累的情况下，P40 mRNA仍处于可翻译状态。第四种化学物质EGTA，一种已知的肌肉细胞融合抑制剂，可阻断肌肉特异性肌动蛋白和原肌球蛋白mRNA的翻译。另一方面，它对P40 mRNA的翻译没有影响。然而，向经EGTA处理的培养物中添加Ca2+，在四天内几乎完全逆转了肌动蛋白原肌球蛋白mRNA翻译的阻断。伴随肌肉mRNA翻译阻断的Ca2+逆转，P40 mRNA进入非翻译状态。因此，观察到管家P40 mRNA的翻译与肌肉特异性mRNA之间呈负相关。在依赖mRNA的微球菌核酸酶处理的同源无细胞提取物中检测了模拟体内P40 mRNA翻译调控的能力。来自成肌细胞和肌管的提取物能够翻译P40 mRNA。此外，含有内源性mRNA的成肌细胞和肌管提取物的核糖体也能够结合P40和肌动蛋白mRNA。然而，肌管提取物对P40 mRNA的结合能力比对肌动蛋白mRNA的结合能力低。通过用高离子强度缓冲液洗涤这些核糖体获得的蛋白质添加后，肌管提取物核糖体结合P40 mRNA的能力有所增强。为了阐明mRNA与蛋白质之间的相互作用在P40 mRNA翻译控制中的作用，还检测了多核糖体和游离P40 mRNA-蛋白质（mRNP）复合物的多肽组成。通过对大鼠L6细胞进行紫外线照射，使多核糖体和游离P40 mRNP复合物的RNA和蛋白质部分共价连接，随后进行杂交选择。对这些复合物浮力密度的分析表明，游离P40 mRNP的蛋白质含量略低于多核糖体P40 mRNP。此外，对游离和多核糖体P40 mRNP复合物的多肽组成分析表明，它们由相同的多肽组成。这些复合物多肽组成之间唯一可检测到的差异是，72kDa和55kDa的两种多肽在多核糖体P40 mRNP中比在游离P40 mRNP中更丰富。
